DC-SIGN ligation on MDDCs induces ERK phosphorylation and collaborates with TNF-α–initiated signals for enhanced ERK activation. (A) Activation of ERK by DC-SIGN engagement. DC-SIGN on MDDCs was engaged by the anti–DC-SIGN MR-1 antibody and the cells were incubated at 37°C for 5 minutes. For comparison, cells were treated with the indicated combinations of antibodies or TNF-α (20 ng/mL). Following cell lysis, phosphorylated ERK and phosphorylated p38 were detected using specific polyclonal antisera. Blots were then stripped and probed for α-tubulin levels as a control for protein loading (bottom panel). (B) DC-SIGN on MDDCs was engaged by the anti–DC-SIGN MR-1 antibody, and the cells were incubated at 37°C for the indicated periods of time. For comparison, cells were treated with an antibody against CD70 or TNF-α (20 ng/mL) for 5 minutes. Following cell lysis, phosphorylated ERK, p38, and Akt, and total ERK were detected using specific polyclonal antisera. Blots were then stripped and probed for α-tubulin levels as a control for protein loading. (C-D) Immature MDDCs from 2 independent donors were incubated with an antibody against DC-SIGN (MR-1) or against CD70, either alone or in combination with TNF-α (20 ng/mL) or LPS (10 ng/mL), and the cells were incubated at 37°C for 5 minutes. For comparison, cells were treated with either LPS (10 ng/mL) or TNF-α (20 ng/mL) for 5 minutes. Following cell lysis, phosphorylated ERK (pERK), phosphorylated p38 (pp38), or total ERK content (ERK) was detected using specific polyclonal antisera. Each experiment was done on MDDCs from at least 4 independent donors, and representative experiments are shown. In all panels, NS refers to cells that were not stimulated.